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1 Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany Therapeutic use of hair follicle-derived epithelial stem cells using.

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Presentation on theme: "1 Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany Therapeutic use of hair follicle-derived epithelial stem cells using."— Presentation transcript:

1 1 Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany Therapeutic use of hair follicle-derived epithelial stem cells using a murine stem cell deficiency model Ewa Anna Meyer-Blazejewska, Hongshan Liu, Mindy K. Call, Ursula Schlötzer-Schrehardt, Winston W-Y. Kao and Friedrich E. Kruse 2 Department of Ophthalmology, University of Cincinnati, OH, USA The authors have no financial interest in the subject matter of this poster Slide 1

2 Introduction: Hair follicle stem cells The bulge region of the hair follicle (HF) is a major reservoir of multipotent adult stem cells (SC). (Cotsarelis et al. 1990) bulge Murine HF Cytokeratin 15 (K15), a marker for stem and progenitor cells in the bulge and outer root sheath of the hair follicle. (Cotsarelis et al., 1990, 1999; Fiqueira et al., 2007) Slide 2

3 K12- Pax6- Hair follicle Cornea outer root sheath inner root sheath bulge Sebaceous gland No expression of the corneal epithelial markers (K12, Pax6) in hair follicle K12+ Pax6+/K12+ Introduction: Hair follicle markers Slide 3

4 Blazejewska et al.; Stem Cells, 2008 Induction of K12 and Pax6 expression in hair follicle SC in vitro using conditioned medium (CM) derived from limbal stroma fibroblasts Cytokeratin 12 molecules K12/molecules ß-actin x10 3 n=5 * * ** Pax 6 central corneal fibroblast CM peripheral corneal fibroblast CM limbal fibroblast CM 3t3 fibroblast CM molecules Pax6/molecules ß-actin x10 3 central corneal fibroblast CM limbal fibroblast CM 3t3 fibroblast CM K12K12/Pax6 peripheral corneal fibroblast CM * * ** Introduction: previous work Slide 4

5 Purpose To explore the therapeutic potential of murine hair follicle-derived stem cells to treat limbal stem cell deficiency and replenish corneal epithelium using an in vivo animal model. Slide 5

6 Method:Tri-Transgenic Mouse Model Inducible K12 driven expression of EGFP K12IRESrtTA Dox rtTA tet-O p minCMV cre rtTA pCAmT mG pCAmG Dox lox P Slide 6

7 We have generated a tri-transgenic mouse model that is both tissue specific and inducible and allows for the detection of K12 expressing cells by the presence of green fluorescence. This transgenic mouse system is comprised of three parts the first of which is the K12 rtTA line that provides the tissue specificity. This line was generated via a knock-in strategy in which an IRES- rtTA (Internal Ribosome Entry Site-reverse tetracycline Transcriptional Activator) minigene was inserted directly after the stop codon of the mouse Krt12 gene. Thereby only differentiated corneal epithelial cells are able to express rtTA. The second component of the tri-transgenic mouse model is Tet-O-Cre. This line uses components of the Tet-On system and together with the K12 rtTA line provides the ability for induction. Specific Tetracycline operator (Tet-O) elements are followed by a CMV min (CMV minimal) promotor and the Cre recombinase gene. In the absence of tetracycline or a tetracycline derivate such as doxycycline, rtTA is unable to bind to the promotor and therefore Cre is not produced. Once doxycycline is added to the system, it can bind with rtTA and together this complex can further bind to the Tet-O elements and drive the expression of Cre. The third component of the system is the ROSA26 mTmG line (Jackson Laboratories) which serves as a dual reporter. This mouse line has loxP sites flanking a membrane-targeted tdTomato (mT) cassette and express red fluorescence in all cell types. Upon breeding to a Cre recombinase mouse line (Tet-O- Cre), the resulting offspring will have td Tomato cassette deleted only in the cells expressing Cre (only in K12 positive cells) allowing for expression of a membrane-targeted enhanced green fluorescent protein (mG). This system allows for the live visualization and tracking of K12 expressing cells. Method:Tri-Transgenic Mouse Model Slide 7

8 Method: Clonal expansion of hair follicle SC Stem cell clones grown on a 3T3 feeder layer Z-stack, 3D Epithelial cells 3T3 cells SC clone K15 SC clones Red fluorescence: no K12 expression in HF-derived epithelial SC clones SC clone K12 rtTA /Tet-O-Cre/ROSA mTmG Slide 8

9 Method: Transplantation of SC on a fibrin gel SC clones subcultured on a fibrin gel as carrierAfter limbal SC debridement Directly after SC transplantation Red: SC and progenitor cells No Green: no K12 expression Fibrin gel K12 rtTA /Tet-O-Cre/ROSA mTmG Slide 9

10 Results: K12 induction post-transplantation 3 days postoperative WT C57/Black6 K12 + cells Regular light Fluorescein staining 14 days postoperative Fibrin gel remains Mouse eye 21 days postoperative Slide 10

11 Results: K12 induction post-transplantation DAPIEGFP tdTomatoMerge Corneal epithelium: 7days postoperative The specimen was prepared by removing the cornea, treating with 0.2% sodium borohydride for 45 min at room temperature (helps in the reduction of background fluorescence), counterstaining with DAPI overnight, and imaging. The total thickness of the Z-stack is 37.5 µm with each slice having a thickness of 1.5 µm. All images are from slice 14 of the Z-stack. K12 + (green) no K12 (red) Slide 11

12 Conclusions Hair follicle bulge-derived epithelial SC possess the potential to differentiate into corneal epithelial-like phenotype in vivo. Hair follicle SC express K12 (corneal epithelial differentiation marker) and regenerate the corneal epithelium up to 3 weeks post-transplantation when transplanted in a murine limbal SC deficiency model. Slide 12


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